| With the improvement of people's living standards,people's demand for livestock and poultry meat products is increasing,so the number of large-scale livestock and poultry farms are increasing fast.However,the discharge of livestock and poultry manure has brought serious environmental problems,especially in the large-scale pig farm with blister manure process.It is difficult to make solid-liquid separation of pig manure after long-time immersion.The high concentration of ammonia nitrogen and chemical oxygen demand(COD)in the waste water make it difficult for the harmless treatment and resource utilization of the waste water.Microbial nest technology or ectopic biological fermentation bed technology is an engineering technology for livestock and poultry fecal-water treatment based on the principle of microbial fermentation.It takes the honeycomb as the basic model,uses sawdust,rice husk and crop straw as the "carbon source",and livestock and poultry fecal-water as the "nitrogen source".A microbial reactor operated at suitable carbon nitrogen ratio is built for absorbing and processing fecal-water by using decomposition and fermentation of microorganisms,which can simultaneously solve the problems of harmless treatment and resource utilization of piggery wastes,and realize no pollution and zero discharge of piggery wastes.It is one of the research hotspots in piggery wastes treatment.Heterotrophic nitrification-aerobic denitrification(HN-AD)microorganisms can realize simultaneous nitrification and denitrification in one reactor under aerobic conditions,which shortens the reaction cycle,saves space area and operation costs,and has strong environmental tolerance.It overcomes the defects of traditional biological denitrification.However,there are few studies on the transcription level of key enzyme genes involved in simultaneous heterotrophic nitrification and aerobic denitrification of HN-AD bacteria,the construction of HN-AD complex flora and its application in bioaugmentation denitrification of wastewater.In this study,by using microbial nest technology to treat the piggery wastes of blister fecal pig farm as an example,through the analysis of the physical and chemical properties,spectral characteristics and microbial community structure of microbial nest pile,this thesis expounds the mechanism of microbial nest to treat fecal sewage,so as to provide theoretical guidance for better maintenance and management of microbial nest in treatment of pig fecal sewage.According to the changes of nitrogen species and the composition of microbial community in the process of microbial nest treatment of piggery wastes,the HN-AD strains were screened to explore their denitrification characteristics and denitrification mechanism.At the same time,the compound flora was constructed and applied to the bioaugmentation denitrification of wastewater treatment,so as to provide a new idea for the treatment of breeding wastewater.The specific research contents and conclusions are as follows:1.Study on physicochemical properties and spectral characteristics of piggery wastes treated by microbial nest technology.Through the study on the changes of material color,odor,temperature,moisture content,pH,conductivity,carbon nitrogen ratio,NH4+-N/NO3--N ratio,E4/E6 and water-soluble heavy metal ions in the process of piggery wastes treatment by microbial nest technology,it is found that the process is a process of continuous decay of organic matter,which can be divided into heating period(1-7 days)and high temperature period(7-84 days)and cooling period(84-112 days).In three different stages,the degradation of organic matter and water evaporation mainly occur in the heating period and high temperature period.In the cooling period,the ability of microbial nest to treat piggery wastes decreases,the water content increases,and the concentration of water-soluble heavy metal ions also increase.From the three-dimensional fluorescence spectrum of microbial nest materials,it can be seen that the fluorescence intensity of proteins is decreasing with the increase of fermentation time.The fluorescence intensity of fulvic acids and humic acids increased continuously,which was consistent with the changes of substances in the reactor,indicating that the treatment of piggery wastes by microbial nest was a process of humification.At the same time,the infrared spectrum of the extract of microbial nest materials showed that amino acids,alcohols,phenols,amides,ketones,lipids and aromatic organic compounds increased.The intensity of each peak is gradually weakened,that is,it is continuously decomposed,which further proves that the process of microbial nest treating piggery wastes is a process of humification.2.Analysis on bacterial community structure of piggery wastes treatment by microbial nest technologyThe V3-V4 hypervariable region of 16S rRNA gene was sequenced and analyzed by Illumina Miseq sequencing platform.Based on the richness,diversity,uniformity and coverage of microbial community in alpha diversity analysis,it is found that species richness,diversity,uniform distribution and coverage are high in high temperature period There were significant differences among Pseudoxanthomonas,Bacillus,Pantoea,Ochrobactrum,Streptomyces,Microbacterium and Enterobacter in the microbial nest at different periods.The dominant bacteria in high temperature period were proteus Bacillus,Streptomyces and Pseudomonas.These bacteria are mainly organic matter and cellulose degrading bacteria.The relevant order of the environmental factors on the bacterial community was moisture content>temperature>C/N>pH>EC>TN>TC>E4/E6>NO3--N>NH4+-N,and the water content has the greatest impact on the bacterial community;At the same time,it was found that the functional abundance of denitrification in high temperature period is relatively high,indicating that there are a large number of denitrifying bacteria in the microbial nest,which convert nitrogen-containing compounds into nitrous oxide or discharge nitrogen.3.Analysis on fungal community structure of piggery wastes treated by microbial nest technologyThe ITS1-ITS2 gene region of fungal was sequenced and analyzed by Illumina Miseq sequencing platform.According to the changes of Ace index,Chaol index,Shannon index,Simpson index,Smithwilson index and Coverage index,the bacterial species were more abundant and diverse in high temperature period than those in heating period and cooling period.The whole fermentation process was mainly Ascomycota and Basidiomycota at the phylum level.Thermophilic fungi such as Talaromyces,Fusarium,Wallemia,Aspergillus,Trichoderma,Thermomyces,Penicillium and Geotrichu at the genus level were abundant in high temperature period,all of which were the dominant bacteria for the degradation of organic substances such as cellulose.The order of environmental factors on fungal community was C/N>moisture content>EC>E4/E6>temperature>TN>TC>NO3--N>NH4+-N>pH.The ratio of carbon to nitrogen had the greatest impact on fungal community.4.Screening of high-efficiency heterotrophic nitrification-aerobic denitrification strains and research on their denitrification characteristicsAccording to the change of total nitrogen in the microbial nest and the prediction of the bacterial function,it is inferred that there are heterotrophic denitrification strains in the microbial nest.Using acetamide as the screening medium for both carbon source and nitrogen source,three highly efficient heterotrophic nitrification strains SDU2,SDU10 and SDU20 were screened from the microbial nest samples.Through physiological and biochemical characteristics,homology comparison of 16S rRNA gene sequences and construction of phylogenetic tree,the strains were identified as Delftia tsuruhatensis,Pseudomonas stutzeri,and Alcaligenes faecalis,respectively.The effects of carbon source,carbon nitrogen ratio,temperature,pH,rotating speed,metal ion concentration and initial ammonia nitrogen concentration on the heterotrophic nitrification characteristics of the screened strains were studied,and the optimum reaction conditions and environmental adaptability were determined.Under aerobic conditions,NO3--N and NO2--N were used as the initial nitrogen source,verifying that the strains SDU2,SDU10 and SDU20 all have aerobic denitrification characteristics.In addition,SNDM1(NH4+-N and NO3--N)and SNDM2(NH4+-N and NO2--N)were used as culture media to verify the characteristics of simultaneous heterotrophic nitrification and aerobic denitrification.Strain SDU2 performed both heterotrophic nitrification of ammonia nitrogen and aerobic denitrification of nitrate nitrogen in SNDM1 and SNDM2 medium,and the total nitrogen removal efficiency was 68.0%and 68.2%,respectively.Strain SDU10 performed both heterotrophic nitrification of ammonia nitrogen and aerobic denitrification of nitrate nitrogen in SNDM1 and SNDM2,and the total nitrogen removal efficiency was 78.7%and 77.9%,respectively.Strain SDU20 is carrying out heterotrophic nitrification at the same time and has another denitrification pathway that does not go through NO3--N and NO2--N.5.iscussion on denitrification mechanism of strain P.stutzeri SDU10By sequencing the whole genome of strain SDU10,the total genome length of strain SDU10 was 4590354 bp and the GC content was 63.88%.The prediction results of coding genes showed that the strain had 4406 coding genes,61 tRNAs,3 rRNAs and 57 sRNAs,accounting for 87.27%of the genome.There are 22 genes related to denitrification metabolism,including ammonia monooxygenase gene(amoA),nitrate reductase gene(napA)and nitrite reductase gene(nirS).The knockout of amoA,napA and nirS genes limited the growth and metabolism of strain SDU10.Compared with wild strains,the knockout strains significantly reduced the removal efficiency of ammonia nitrogen,nitrate nitrogen and nitrite nitrogen,which were 8.5%,18.5%and 21.6%,respectively.At the same time,the knockout of genes reduced the transcription of related genes,which further verified the importance of these three genes.6.Construction of HN-AD complex flora and study on bioaugmentation denitrification.Firstly,when SDU2,SDU10 and SDU20 were used to treat piggery wastewater at the shake flask level,the removal efficiency of NH4+-N and COD were higher with carbon source addition than those without additional carbon source,which verified that the screened strains had the potential to be used in wastewater treatment,especially with carbon source addition.Secondly,through orthogonal experiment,the compound flora with the optimal initial addition volume ratio of strains SDU2,SDU10 and SDU20 of 2:3:3 was constructed.The bioaugmentation denitrification removal experiment was carried out by using the composite flora.In the experimental group,the COD and NH4+-N in the SBR1 reactor decreased rapidly,and the removal efficiency were 93.7%and 88.9%respectively.In the control group,the removal efficiency of COD and NH4+-N in SBR2 reactor were 75.8%and 68.4%respectively,indicating that the addition of compound flora can effectively remove the concentrations of COD and NH4+-N in the reactor.At the same time,the community structure of the two reactors changed after the addition of compound bacteria in SBR1 reactor.Alcaligenes,Pseudomonas and Paracoccus in SBR1 are the dominant species,with higher abundance than that in SBR2 reactor.The addition of compound bacteria changed the bacterial structure,and the aerobic denitrifying bacteria gradually increased.At the same time,combined with the changes of ammonia nitrogen concentration and COD concentration in the effluent of SBR1 reactor,it is speculated that the addition of compound denitrification bacteria is conducive to the adjustment of sludge bacteria structure and the completion of bioaugmentation denitrification. |
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